Bacillus licheniformis with anti-inflammatory and antibacterial effects and application thereof

Abstract

The application discloses a bacillus licheniformis YL-001 with anti-inflammatory and antibacterial effects and an application thereof, and belongs to the technical field of biotechnology. The bacillus licheniformis YL-001 is separated from soil. The preservation number of the bacillus licheniformis is GDMCC No.66556. The metabolic product of the bacillus licheniformis YL-001 provided in the application has the abilities of significantly inhibiting secretion of inflammatory factors and growth of streptococcus pneumoniae and hemolytic streptococcus b, and can improve the total antioxidant capacity of serum. The strain has good biocompatibility and high safety, has important significance for development of anti-inflammatory or antibacterial drugs, health products and food, and is especially suitable for preparation of products with throat-protecting effects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a strain of Bacillus licheniformis with anti-inflammatory and antibacterial effects and its applications. Technical Background

[0002] Pharyngitis is a common upper respiratory tract infection, mainly caused by pathogenic microorganism infection, environmental irritation, or weakened immunity. Symptoms include redness, swelling, pain, dryness, and itching in the throat. Currently, treatment for pharyngitis primarily relies on antibiotics or anti-inflammatory drugs, but long-term use may lead to increased drug resistance or significant side effects. Furthermore, while traditional Chinese medicine has some efficacy, its effects are slow, and some products have complex ingredients and are difficult to control in terms of quality.

[0003] In recent years, probiotics and their metabolites have attracted attention due to their potential anti-inflammatory, antibacterial, and immunomodulatory functions. Bacillus licheniformis, a common probiotic, has been shown to regulate gut microbiota and enhance immunity. However, the metabolites of existing strains have limited efficacy in inhibiting the secretion of inflammatory factors, antioxidant capacity, and inhibitory effects against common throat pathogens (such as Streptococcus pneumoniae and beta-hemolytic streptococci), making it difficult to meet the clinical need for high-efficiency and safe treatment.

[0004] Therefore, there is an urgent need to develop a strain of Bacillus licheniformis with significant anti-inflammatory and antibacterial effects and high biocompatibility, and to explore its application in the preparation of throat-clearing and pharyngeal-soothing products, so as to provide a safer and more effective solution for the treatment of related diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Bacillus licheniformis with anti-inflammatory and antibacterial effects and its applications. This invention isolates and screens a strain of Bacillus licheniformis YL-001 from soil. The metabolites obtained from its fermentation not only have significant anti-inflammatory effects, but also have a good ability to inhibit the growth of Streptococcus pneumoniae and beta-hemolytic streptococci. This strain is of great significance for the preparation of anti-inflammatory or antibacterial drugs, health products, and foods, and is especially suitable for the preparation of products with throat-soothing effects.

[0006] Physicochemical analysis and 16S rRNA gene alignment identified the strain with anti-inflammatory and antibacterial effects as *Bacillus licheniformis*, named *Bacillus licheniformis* YL-001. This *Bacillus licheniformis* strain was deposited on June 19, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 66556), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] In vitro experiments demonstrated that the metabolites of this Bacillus licheniformis strain did not exhibit significant cytotoxicity against RAW264.7 cells; and that it significantly inhibited the secretion of interleukin-6 (IL-6), interleukin-8 (IL-8), nitric oxide (NO), and tumor necrosis factor-α (TNF-α) by RAW264.7 cells, demonstrating significant anti-inflammatory effects; and that it also showed good inhibitory effects against Streptococcus pneumoniae and beta-hemolytic streptococci.

[0008] Rat experiments have shown that the metabolites obtained by fermentation of this Bacillus licheniformis have a median lethal dose (LD50) greater than 10.00 g / kg·bw in SD rats; and can significantly reduce the levels of inflammatory factors (TNF-α, IL-6) and the content of the inflammatory marker CRP in the serum of rats with chronic pharyngitis induced by beta-hemolytic streptococcus, and significantly increase the total antioxidant capacity (T-AOC) in the serum.

[0009] Preferably, the preparation of the metabolite of Bacillus licheniformis YL-001 includes the following steps:

[0010] S1: Inoculate Bacillus licheniformis YL-001 into TSB liquid medium and culture until the OD value is 2-3 to obtain the inoculum solution;

[0011] S2: Inoculate the bacterial culture solution into the fermentation medium at 1-3 v / v%, ferment for 24-36 h, filter, and obtain the fermentation filtrate;

[0012] S3: After sterilizing the fermentation filtrate, freeze-dry it to obtain the metabolites.

[0013] Preferably, the fermentation medium is TSB liquid medium;

[0014] More preferably, the fermentation medium is a self-made liquid culture medium;

[0015] Furthermore, the self-made liquid culture medium is composed of TSB liquid culture medium and plant compound powder;

[0016] Furthermore, the plant compound powder accounts for 2 wt% of the self-made liquid culture medium;

[0017] Furthermore, the plant compound powder is composed of amla powder, platycodon powder, and chebula powder in a mass ratio of 1:1-4:6-8.

[0018] Another object of the present invention is to provide the application of Bacillus licheniformis YL-001 in the preparation of products with throat-soothing effects.

[0019] Preferably, the product is a medical preparation.

[0020] Preferably, the medical preparation is any one of tablets, sprays, oral liquids, capsules, or granules.

[0021] Preferably, the medical preparation further includes excipients acceptable for medical preparations.

[0022] Beneficial effects of this invention:

[0023] In vitro experiments demonstrated that Bacillus licheniformis YL-001 did not exhibit significant cytotoxicity against RAW264.7 cells; the metabolites obtained from the fermentation of this Bacillus licheniformis (Application Example 1) showed significant anti-inflammatory effects on the secretion of NO, IL-6, and IL-8 by RAW264.7 cells; and the metabolites of this Bacillus licheniformis showed good inhibitory effects on Streptococcus pneumoniae and beta-hemolytic streptococci; furthermore, this invention further discovered that adding 2 wt% of the mass ratio of Bacillus licheniformis YL-001 to the fermentation medium of Bacillus licheniformis YL-001... The plant mixture powder composed of Phyllanthus emblica powder, Terminalia chebula powder, and Platycodon grandiflorus powder in a 1:2:7 ratio can further enhance the bioactivity of Bacillus licheniformis YL-001 fermentation product. The metabolites obtained by fermenting and culturing Bacillus licheniformis as the fermentation strain in the self-made liquid culture medium of the present invention (Application Example 2) showed a significant increase in the inhibition rate of NO, IL-6, and IL-8 secretion by RAW264.7 cells compared with Application Example 1; and compared with Application Example 1, it also had a better inhibitory effect on Streptococcus pneumoniae and beta-hemolytic streptococci.

[0024] Rat experiments demonstrated that the metabolite obtained from the fermentation of *Bacillus licheniformis* had a median lethal dose (LD50) greater than 10.00 g / kg bw in SD rats. Furthermore, it significantly reduced the levels of inflammatory factors in the serum of rats with beta-hemolytic streptococcal-induced chronic pharyngitis, particularly TNF-α, IL-6, and the inflammatory marker CRP, while significantly increasing the total antioxidant capacity (T-AOC) of the serum. In addition, compared to Application Example 1, Application Example 2 further enhanced the reduction rate of TNF-α, IL-6, and the inflammatory marker CRP levels in the serum of rats with beta-hemolytic streptococcal-induced chronic pharyngitis, as well as the increase in the total antioxidant capacity (T-AOC) of the rat serum.

[0025] Based on the above experimental data, Bacillus licheniformis YL-001 has the following significant effects:

[0026] (1) It has good biocompatibility with RAW264.7 cells;

[0027] (2) Significantly inhibits the secretion of IL-6, IL-8 inflammatory factors and NO signaling molecules by RAW264.7 cells;

[0028] (3) It has an inhibitory effect on both Streptococcus pneumoniae and beta-hemolytic streptococcus;

[0029] (4) The median lethal dose for SD rats is greater than 10.00 g / kg·bw;

[0030] (5) It can significantly reduce the levels of TNF-α, IL-6 and CRP in the serum of rats with chronic pharyngitis induced by beta-hemolytic streptococcus, and significantly increase the level of T-AOC in the serum of rats.

[0031] Preservation of biological materials

[0032] A strain of Bacillus licheniformis, YL-001, was deposited on June 19, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 66556), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments. It should be noted that these descriptions of embodiments are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0034] Unless otherwise specified, all materials and reagents used in the examples are commercially available.

[0035] The culture medium involved in this invention:

[0036] MRS liquid culture medium: 10.0 g / L peptone, 5.0 g / L beef extract, 4.0 g / L yeast extract, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 20.0 g / L glucose, 1.0 g / L Tween 80, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, sterilized at 121℃ for 15 min, pH 6.2±0.2.

[0037] TSB liquid medium: 17.0 g / L tryptone, 3.0 g / L soybean papain hydrolysate, 2.5 g / L dipotassium hydrogen phosphate, 5.0 g / L sodium chloride, 2.5 g / L glucose, sterilized at 121℃ for 15 min, pH 7.3±0.2.

[0038] Homemade liquid culture medium: 17.0 g / L tryptone, 3.0 g / L soybean papain hydrolysate, 2.5 g / L dipotassium hydrogen phosphate, 5.0 g / L sodium chloride, 2.5 g / L glucose, 2 g / L amla powder, 4 g / L green plum powder, 14 g / L platycodon powder, sterilized at 121℃ for 15 min, pH 7.3±0.2.

[0039] Example 1: Isolation, Screening and Identification of Strains

[0040] 1. Discovery and isolation of strains

[0041] Materials: Soil samples from the green fruit planting base. Take 5g of soil and place it in 50mL of sterile physiological saline and stir evenly to obtain a soil suspension.

[0042] Take 5 mL of soil suspension and add it to 45 mL of sterile physiological saline. Shake well, then take 0.1 mL and drop it onto MRS solid medium, spreading it evenly. Invert the plate and incubate at 37°C for 1 day. Select colonies with significant differences and streak them onto MRS solid medium for isolation. Incubate at 37°C for 1 day. After repeating the streaking process 3 times, 4 single colonies (strain 1-strain 4) were isolated and purified. Single colonies were selected for inflammatory factor inhibition assay to detect the changes in the inhibition rate of TNF-α by the metabolites of strains 1-4. Finally, one strain that can significantly inhibit TNF-α was selected and labeled as strain 1.

[0043] The preparation steps for the metabolites of bacteria 1-4 to be tested are as follows:

[0044] Step 1: After activating the bacteria to be tested, dilute it with sterile water to a concentration of 1×10⁻⁶. 7 CFU / mL bacterial culture solution;

[0045] Step 2: Inoculate the bacterial culture solution into MRS liquid medium at an inoculation rate of 2 v / v%, ferment for 24 h, and then filter to obtain the fermentation filtrate;

[0046] Step 3: Freeze-dry the fermentation filtrate and sterilize it by irradiation to obtain the metabolites.

[0047] Sample preparation: The metabolites of bacteria 1-4 were prepared into 10 wt% test sample solutions using DMEM medium as the solvent.

[0048] Cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. RAW264.7 cells in logarithmic growth phase were seeded into 24-well plates, with a cell count of 1 × 10⁵ cells per well. After 24 hours of incubation, the supernatant was discarded, and the cells were divided into model, control, and experimental groups, with three replicates per group. 0.5 ml of experimental sample was added to each experimental group, while equal volumes of DMEM medium were added to the control and model groups. LPS was then added to both the model and experimental groups to achieve a final concentration of 1 μg / ml, while no LPS was added to the control group. After 24 hours of induction, the cell supernatant was collected, and the content of the inflammatory factor TNF-α in the LPS-induced cell supernatant was measured using an ELISA kit.

[0049] Table 1. Screening of bacterial strains that inhibit TNF-α secretion by RAW264.7 cells.

[0050] Group TNF-α content (pg / mL) Blank group 281.09±5.25 Model group <![CDATA[754.82±10.37 # ]]> Bacteria 1 527.44±7.51* Bacteria 2 694.76±15.42* Bacteria 3 731.38±10.15 Bacteria 4 685.71±11.94*

[0051] Note: "*" indicates that p < 0.05 compared to the model control group. # "" indicates that compared with the blank group, p < 0.05.

[0052] Based on the results of TNF-α secretion by RAW264.7 cells, strain 1 was selected as the optimal strain for preservation.

[0053] Strain morphological identification

[0054] After plate culture, strain 1 was morphologically identified and Gram-stained. This bacterium is Gram-positive, and its colonies are dirty white with irregular edges, dry and wrinkled surfaces. Oval terminal spores can be seen after staining.

[0055] molecular biological identification of strains

[0056] The selected strain 1 was identified using molecular biological identification methods, and sequencing revealed that its 16S rRNA is shown in SEQ ID NO. 1.

[0057] .

[0058] The sequencing results were compared with the NCBI database using BLAST and a phylogenetic tree was constructed. The results showed that the sequence of this strain was highly homologous to the gene sequence of Bacillus licheniformis, with a homology of 100%. Based on its morphological characteristics, strain 1 was identified as belonging to Bacillus licheniformis.

[0059] The strain was named Bacillus licheniformis YL-001 and deposited on June 19, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, postal code 510070, with accession number GDMCC No:66556.

[0060] Preparation of metabolites from Bacillus licheniformis YL-001: The preparation method includes the following steps:

[0061] Strain activation: Bacillus licheniformis YL-001 was inoculated into TSB liquid medium and activated until the OD value was 2 to obtain a strain solution. The strain solution was vortexed and mixed to obtain a YL-001 bacterial suspension.

[0062] Application Example 1:

[0063] Step 1: Dilute the activated YL-001 bacterial suspension with sterile water to a concentration of 1×10⁻⁶. 7 CFU / mL bacterial culture solution;

[0064] Step 2: Inoculate the bacterial culture solution into TSB liquid medium at an inoculation rate of 2 v / v%, ferment for 36 h, filter to obtain fermentation filtrate, and add sterilized fresh TSB liquid medium once after 18 h of fermentation, with the amount of replenishment being 20 v / v% of the original TSB liquid medium.

[0065] Step 3: Freeze-dry the fermentation filtrate and sterilize it by irradiation to obtain the metabolites.

[0066] Application Example 2:

[0067] Step 1: Dilute the activated YL-001 bacterial suspension with sterile water to a concentration of 1×10⁻⁶. 7 CFU / mL bacterial culture solution;

[0068] Step 2: Inoculate the bacterial culture solution into the self-made liquid culture medium at an inoculation rate of 2v / v%, ferment for 36 hours, filter to obtain fermentation filtrate, and add sterilized fresh self-made liquid culture medium once after 18 hours of fermentation, with the amount of replenishment being 20v / v% of the original self-made liquid culture medium.

[0069] Step 3: Freeze-dry the fermentation filtrate and sterilize it by irradiation to obtain the culture product.

[0070] Comparative application example 1:

[0071] Unlike Application Example 1, Bacillus licheniformis King58 with accession number CGMCC No.29319 was used to replace Bacillus licheniformis YL-001 for fermentation, while the other parameters and steps were the same as in Application Example 1.

[0072] Compare with application example 2:

[0073] Unlike Application Example 1, Bacillus licheniformis HCD02 with accession number CGMCC No. 31449 was used to replace Bacillus licheniformis YL-001 for fermentation, while the other parameters and steps were the same as in Application Example 1.

[0074] Efficacy verification:

[0075] Sample preparation for experiments 1 and 2: Take application examples 1-2 and control application example 1 and prepare them into 10 wt% mixtures using DMEM culture medium, and seal them for later use.

[0076] Test 3 Sample preparation: Take Application Example 1-2 and Comparative Application Example 1-2 and prepare 10wt% mixed solutions using sterile deionized water, and seal them for later use.

[0077] Experiment 4 Sample preparation: Take the samples from Application Examples 1-2 and prepare them into 25 wt% mixtures using physiological saline, then seal and set aside for use.

[0078] Experiment 5 Sample preparation: Take the samples from Application Examples 1-2 and prepare them into 10wt% mixtures using physiological saline, then seal and set aside for use.

[0079] Experiment 1: Detection of Bacillus licheniformis cytotoxicity

[0080] Cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. RAW264.7 cells in logarithmic growth phase were seeded in 96-well plates, with a cell count of 8000 per well. After 24 hours of incubation, the supernatant was discarded. 100 μL of experimental sample was added to each test well, and an equal volume of DMEM medium was added to each control well. Blank wells (containing only DMEM medium without RAW264.7 cells) were also included. All control, test, and blank wells were prepared in triplicate. After another 24 hours of incubation, cell viability was measured using the CCK-8 assay. Results are expressed as mean ± standard deviation (two decimal places).

[0081] Table 2. Results of cytotoxicity assays of metabolites on RAW264.7 cells.

[0082] Group Cell viability / % control group 100 Application Example 1 96.52±1.41 Application Example 2 95.47±1.93 Comparative Application Example 1 95.28±1.46

[0083] As shown in Table 2, the application examples 1-2 and comparative application example 1 provided by this invention do not exhibit significant cytotoxicity.

[0084] Experiment 2: Inhibition of inflammatory factors by Bacillus licheniformis metabolites

[0085] Cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. RAW264.7 cells in logarithmic growth phase were seeded into 24-well plates, with a cell count of 1 × 10⁵ cells per well. After 24 hours of incubation, the supernatant was discarded, and the cells were divided into model, control, and experimental groups, with three replicates per group. 0.5 ml of experimental sample was added to each experimental group, while equal volumes of DMEM medium were added to the control and model groups. LPS was then added to both the model and experimental groups to achieve a final concentration of 1 μg / ml, while no LPS was added to the control group. After 24 hours of induction, the cell supernatant was collected. The NO content in the cell supernatant was measured at 540 nm using a microplate reader using a NO detection kit. The levels of inflammatory factors (IL-6 and IL-8) in the cell supernatant after LPS induction were measured using an ELISA kit. Results are expressed as mean ± standard deviation (two decimal places).

[0086] Table 3 Results of Inflammatory Factor Inhibition

[0087]

[0088] Note:" # "" indicates that p < 0.05 compared to the blank group; "*" indicates that p < 0.05 compared to the comparison application example 1.

[0089] As shown in Table 3, the inflammatory factors in the model control group were significantly increased compared with the blank group, indicating that the model was successfully established.

[0090] The results of Application Example 1 and the model control group show that Bacillus licheniformis YL-001 provided by the present invention has the effect of inhibiting the secretion of cellular inflammatory factors (IL-6, IL-8) and NO signaling molecules.

[0091] The results of Application Examples 1-2 show that the metabolites of Bacillus licheniformis YL-001 provided by this invention, when cultured in the self-made liquid culture medium specified by this invention, have a more significant effect on inhibiting the secretion of cellular inflammatory factors (IL-6, IL-8) and NO signaling molecules.

[0092] A comparison of the results of Application Example 1 and Comparative Application Example 1 shows that, compared with other Bacillus licheniformis with similar effects, the Bacillus licheniformis YL-001 provided by the present invention has a more significant effect in inhibiting the secretion of NO and inflammatory factors by RAW264.7 cells.

[0093] Experiment 3: Verification of the inhibitory efficacy of Bacillus licheniformis YL-001 metabolites against Streptococcus pneumoniae and beta-hemolytic streptococci.

[0094] Preparation of drug sensitivity tablets:

[0095] Qualitative filter paper discs with a diameter of 6 mm were sterilized by autoclaving in a petri dish and then immersed in five different test samples. The negative control group was immersed in sterile deionized water, and the positive control group was immersed in tetracycline (2.5 mg / mL). After immersion for 2 hours, they were air-dried for later use.

[0096] Test method:

[0097] Take 1 mL, concentration 10 8 Suspensions of the test bacteria (Streptococcus pneumoniae and β-hemolytic streptococci) at a concentration of 1 / mL were inoculated onto 5% brain heart broth agar, spread evenly, and then the plates were dried at room temperature for 3-5 minutes. The antimicrobial susceptibility test strips were then gently pressed onto the surface of the agar using tweezers to ensure complete contact. The plates were inverted and incubated at 37°C for 24 hours. The diameter of the inhibition zone was measured using calipers, and the measurement was repeated three times. The average diameter of the inhibition zone was calculated. Judgment criteria: diameter >18 mm was high sensitivity; diameter 12-18 mm was moderate sensitivity; diameter 7-12 mm was low sensitivity; and <7 mm was insensitive. The experiment was performed in triplicate, and the results are expressed as mean ± SE (retaining two decimal places).

[0098] Table 4 Antibacterial test of Bacillus licheniformis YL-001

[0099]

[0100]

[0101] Note: "-" indicates that no inhibition zone was produced.

[0102] The results showed that the metabolites of Bacillus licheniformis YL-001 had a strong inhibitory effect on Streptococcus pneumoniae and beta-hemolytic streptococcus, two common pathogens causing pharyngitis. The metabolites of Bacillus licheniformis cultured in the self-made culture medium of this invention had an even stronger inhibitory effect on Streptococcus pneumoniae and beta-hemolytic streptococcus. No inhibition zone was produced in Comparative Application Examples 1-2. Therefore, Bacillus subtilis in Comparative Application Examples 1-2 had no inhibitory effect on Streptococcus pneumoniae and beta-hemolytic streptococcus.

[0103] Test 4: Acute toxicity test:

[0104] Acute toxicity tests were conducted on the test samples to verify their safety.

[0105] Experimental Methods: Sixty male SD rats weighing 220g ± 20g were selected and divided into two groups. Each group received the test sample orally twice daily at 6-hour intervals, with each gavage volume being 20mL / kg body weight (equivalent to a dose of 10.00g / kg body weight). Rats were fasted for 6 hours before the first gavage. Rats were observed for two weeks after gavage, and the signs of poisoning and mortality were recorded.

[0106] Experimental Results: No obvious symptoms of poisoning were observed in SD rats after gavage, and no deaths were observed during the 14-day observation period. At the end of the observation period, the test animals were euthanized and dissected. No obvious abnormalities were found in the major organs such as the liver, spleen, kidneys, intestines, heart, and lungs. The median lethal dose (LD50) for SD rats in Examples 1-2 of this invention was greater than 10.00 g / kg·bw. According to the acute toxicity dose grading standard in GB15193.3-2014, it belongs to the practically non-toxic category.

[0107] Experiment 5: Protective effect against beta-hemolytic streptococcal-induced chronic pharyngitis in rats

[0108] Experimental Methods: Eighty male SD rats weighing 220g ± 20g were randomly divided into four groups: a blank control group, a model group, and two experimental groups (Examples 1-2). During days 1-15, the model group and experimental group were inoculated with a concentration of approximately 10... 8 Beta-hemolytic streptococci (CFU / mL) were administered at 0.02 mL per rat every three days. The inoculation method was as follows: using sterile forceps, the rat's maxillary buccal mucosa was exposed by retracting it, and a sterile needle was used to puncture the mucosa with beta-hemolytic streptococci at a near-horizontal angle. The blank control group was not inoculated with beta-hemolytic streptococci but only with sterile broth culture medium. The inoculation method, dosage, and frequency were consistent with the model group. Starting from day 16, experimental samples were administered via gavage for four consecutive weeks. The experimental sample dosage was 10 mL / kg body weight. The model group and blank control group were administered the same volume of physiological saline via gavage. Twenty-four hours after the last administration, blood was collected from the orbital venous plexus of each rat, serum was separated, and the levels of TNF-α, IL-6, CRP, and T-AOC in the serum were measured. All detection methods strictly followed the ELISA kit instructions. Experimental results are expressed as mean ± standard deviation (retaining two decimal places).

[0109] Table 5. Serum inflammatory factor levels in rats with a chronic pharyngitis model induced by group A beta-hemolytic streptococcus.

[0110] Group TNF-α (ng / L) IL-6 (pg / ml) CRP (ng / mL) T-AOC (U / mL) Blank control group 146.28±1.91 47.82±4.17 407.11±16.35 52.05±3.38 Model group <![CDATA[263.23±1.50 # ]]> <![CDATA[94.68±3.94 # ]]> <![CDATA[795.16±12.60 # ]]> <![CDATA[25.41±2.30 # ]]> Application Example 1 182.49±4.62* 63.51±7.37* 502.18±10.02* 41.40±4.07* Application Example 2 157.71±3.47* 51.98±6.05* 447.65±9.44* 46.25±3.04*

[0111] Note: "*" indicates that p < 0.05 compared to the model control group. # "" indicates that compared with the blank group, p < 0.05.

[0112] As shown in Table 5, compared with the blank group, the levels of TNF-α, IL-6, and CRP in the model control group were significantly increased, while the level of T-AOC was significantly decreased, indicating that the model was successfully established.

[0113] The results of Application Example 1, compared with those of the model control group, show that the metabolites of Bacillus licheniformis YL-001 provided by this invention have the effects of inhibiting the secretion of TNF-α, IL-6, and CRP and increasing T-AOC.

[0114] The results of Application Examples 1-2 show that the metabolites of Bacillus licheniformis YL-001 provided by this invention, when cultured in the special culture medium specified by this invention, have a more significant effect on inhibiting the secretion of TNF-α, IL-6, and CRP and increasing T-AOC.

[0115] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus licheniformis YL-001, characterized in that, The bacteria were deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 19, 2025, with accession number GDMCCNO: 66556.

2. A microbial agent, characterized in that, Includes Bacillus licheniformis YL-001 as described in claim 1.

3. A fermentation product, characterized in that, It contains Bacillus licheniformis YL-001 as described in claim 1 or the bacterial agent as described in claim 2.

4. The use of Bacillus licheniformis YL-001 as described in claim 1, the bacterial agent as described in claim 2, or the fermentation product as described in claim 3 in the preparation of throat-soothing products.

5. The application according to claim 4, characterized in that, The product is a medical preparation.

6. A medical preparation with throat-soothing effects, characterized in that, It contains at least one of Bacillus licheniformis YL-001 as described in claim 1, the inoculum as described in claim 2, or the ferment as described in claim 3.

7. The medical preparation according to claim 6, characterized in that, The medical preparation is any one of tablets, sprays, oral liquids, capsules, or granules.

8. The medical preparation according to claim 6, characterized in that, It also includes pharmaceutical-acceptable excipients.

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